Background
Protein phosphatase 2A (PP2A) is a member of phosphoprotein phosphatase (PPP) family which comprises cellular serine/threonine phosphatases [
1‐
3]. Actually, decreased activity of PP2A has been reported as a recurrent alteration in many types of cancer [
4]. Moreover, several cellular inhibitors of PP2A have been identified in a variety of cancer types [
3,
5]. CIP2A as a PP2A inhibitor is overexpressed in many human malignancies [
3]. However, FTY720 as a PP2A activator could possess potent antitumor properties via restoration of PP2A activity [
6]. Ceramides as another PP2A activator belong to structural components of the cell membrane, which have potent signaling properties that result in cell apoptosis, senescence, or cell-cycle arrest [
7‐
9]. In addition, PP2A as a tumor suppressor negatively regulates many proliferative signaling pathways associated with cancer progression by dephosphorylating crucial proteins in these pathways such as Wnt/β-catenin, PI3K/Akt and ERK/ MAPK signaling pathway [
4,
10,
11].
Nitric oxide (NO), a major signaling molecule, is involved in various physiological and pathological processes. High level of NO has the cytotoxic and apoptosis-inducing effects on oncogenesis. NO is often derived from both the endogenous way by stimulating NO syntheses and the exogenous way through NO donor [
12]. O
2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K,C
13H
16N
6O
8) is a diazeniumdiolate-based NO donor and is highly cytotoxic to several types of human cancer cells, such as acute lymphoblastic leukemia [
13], hepatocellular carcinoma [
14], prostate cancer cells [
15] or murine erythroleukemia cells [
16]. Moreover, JS-K as a lead NO donor selectively exhibits antitumor effects towards cancer cells while has no significant toxicity toward normal cells [
17]. The nonobese diabetic-severe combined immune deficient mice intravenously injected with JS-K had not display significant hypotension [
18]. Simultaneously, JS-K also inhibited the growth and induced apoptosis of tumor cell lines through different signaling pathway. Ren [
19] demonstrated that JS-K inhibited Hep 3B hepatoma cell growth and induced c-Jun phosphorylation via multiple MAP kinase pathways. JS-K also has been shown to inhibit the prostate cancer cells growth, which could be attributed to inhibit WNT- and AR-signaling via NO-release [
20]. Furthermore, NO also induced cell apoptosis through increasing the level of ceramides [
21], which could increase the activity of PP2A.
Therefore, some signaling pathways such as Wnt/β-catenin, MAPK and Bcl-2 family are meeting points between JS-K and PP2A. In our previous study, JS-K inhibited the proliferation of HepG2 cells and significantly induced apoptosis via Ca
2+/caspase-3-mediated pathway [
22]. The present study demonstrated that the anti-proliferative and apoptosis- inductive effects of JS-K were attributed to increase the activity of PP2A, which caused dephosphorylation of its downstream responsive substrates like β-catenin, c-Myc, and Bcl-2. Furthermore, JS-K increased Bax-to-Bcl-2 ratio, released Cyt c from mitochondria and increased the activities of cleaved-caspase-9/3.Therefore, PP2A activation mechanism contributes to JS-K induced caspase-dependent apoptosis in human hepatocellular carcinoma cells.
Methods
Reagents and antibodies
JS-K, PP2A-Cα (siRNA) and control siRNA-A, antibody against to PP2A-A (α/β), PP2A-B55, PP2A-C (α/β) and p-Bcl-2(Ser70) were purchased from Santa Cruz Biotechnology (San Diego, CA). JS-K was dissolved in 100% DMSO to a concentration of 10 mM as a stock solution. The final concentration of DMSO did not exceed 0.1% throughout the study. Reagents used in the present study included cell counting kit-8(CCK-8) (Dojindo Laboratories, Kumamoto, Japan), Annexin V-FITC/PI kit (BD Biosciences, NJ, San Diego, CA, USA), DAPI staining solution, 3-Amino, 4-aminomethyl-2′,7′-difluorescein, diacetate (DAF-FM DA), 5,5′,6,6′-tetrachloro-1,1′,3,3′- tetraethylbenzimidazolcarbocyanine iodide (JC-1), Carboxy-PTIO, cell lysis buffer kit for Western, enhanced BCA Protein Assay Kit (Beyotime, Haimen, China). Human Protein phosphatase-2A (PP2A) Elisa kit was supplied with Jianglai Institute of Biotechnology (Shanghai, China). Okadaic acid (OA), Z-VAD (OMe)-FMK and antibodies for Bax, Bcl-2, cytochrome c, pro-caspase-3, cleaved-caspase-3, pro-caspase-9, cleaved caspase-9, PARP, cleaved PARP, XIAP, β-catenin, c-Myc, β-actin and COX IV were supplied from Cell Signaling Technology (Beverly, MA, USA). HRP-conjugated affinipure goat anti-mouse IgG and HRP-conjugated affinipure goat anti-rabbit IgG were supplied from Proteintech Group, Inc. (Wuhan, China). FTY720 was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). PPP2CA cDNA (PP2A-Cα, GV230 carrier, NheI/AgeI enzymatic cutting) was purchased from Shanghai Genechem Co.,Ltd.
Cells culture
The human hepatocellular carcinoma cells (HCC) (PLC5, Huh-7, Bel-7402, SMMC-7721 and HepG2) were purchased from Shanghai Institute of Cell Biology (Shanghai, China). PLC5, Bel-7402, and SMMC-7721 cells were maintained in RPMI 1640 medium (Gibco, Invitrogen) while Huh-7 and HepG2 cells were cultured in high glucose DMEM (Gibco, Invitrogen). Both medium were supplemented with 10% FBS. Penicillin 100 U/mL and streptomycin 100 μg/mL were added to the cultured medium. All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2.
Animals
A total of 32 eight-week-old male Wistar rats, weighing 180 ± 10 g, were acquired from Experimental Animal Center of Medical College in Henan University of Science and Technology (Luoyang, China). Animals were acclimatized under standardized conditions (23 ± 2 °C, 60 ± 10% humidity, 12 h light/dark cycle) for one week prior to use. All experimental procedures were approved by and performed in accordance with the guidelines set out by the Institutional Animal Experiment Committee of Henan University of Science and Technology, China.
Cell counting Kti-8 (CCK-8) proliferation assays
The cells at a final density of 1 × 104 cells/well were seeded into 96-well cell plates overnight and incubated with various concentrations of JS-K for 24, 48 or 72 h. Thereafter, the medium with JS-K was removed and rapidly replaced with 100 μL medium containing 10 μL CCK-8 reagent. After incubation at 37 °C for 2 h, the absorbance was measured using a spectrophotometer (Tecan, Switzerland) at a wavelength of 450 nm. Experiments were conducted in triplicate. Inhibition rate (%) = [(OD Control –OD Treated)/ OD Control] × 100%.
DAPI staining and crystal violet staining
HCC cells (5 × 105cells) were seeded into 6-well cell plate for 12 h and then treated with JS-K for 24 h. The typical morphology of cells was confirmed by phase contrast microscopy. Then the cells were washed with PBS twice, and incubated with DAPI in accordance with the manufacturer’s instructions. After staining, the cells were immediately observed by a fluorescence microscope (Olympus, IX-70, Japan). In addition, the cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet while they were exposed to JS-K for 24 h and observed by phase contrast microscopy.
Apoptosis detection
The cells were treated with different concentrations of JS-K for 24 h. In addition, cells were pre-treated with indicated concentration of Carboxy-PTIO (50 μM), OA (1 nM) or FTY720 (2.5 μM) for 1 h, and then the cells were treated with JS-K for indicated time. Thereafter, the cells were collected and resuspended in 500 μL binding buffer after treatment with JS-K. Five microliters Annexin-V -FITC and 5 μL PI were then added to these cells, which were kept in the dark for 10 min. The stained cells were analyzed by flow cytometry and calculated by CellQuest software.
Measurement of mitochondrial membrane potential
Quantitative changes of mitochondrial membrane potential(MMP)at the early stage of the cell apoptosis were measured by JC-1 probe. The cells were treated with different concentrations of JS-K for 24 h and then the cells were collected and resuspended in medium containing JC-1 reagent according to the manufacturer’s instructions. Relative fluorescence intensity was monitored by flow cytometry with excitation source at 530 nm and emissions at 585 nm.
Nitric oxide generation detection
DAF-FM DA as a fluorescent indicator of intracellular NO was also used. The cells were plated at a final density of 5 × 105 cells/well in 6-well cell plate. After treatment with JS-K for 24 h, cells were collected and resuspended with medium containing DAF-FM DA (5 μM) for 20 min at room temperature in the dark. DAF-FM fluorescence intensity was measured by fluorescence microscope and flow cytometry, respectively.
PP2A enzymatic activity assay
The PP2A concentration was measured by Human PP2A ELISA Kit. After treatment with JS-K, the cells were collected and diluted with PBS (pH 7.2–7.4) at a density of 1 × 106 /mL. Then the cells were repeated freeze−thaw cycles three times in order to release of intracellular components. The concentration of PP2A in each supernatant was measured after the cells were centrifuged at 2000–3000 rpm for 20 min and measured the optical density (OD) at 450 nm according to the manufacturer’s instructions.
DNA transfection
Silencing of PP2A: Human PP2Ac small interfering RNA (siRNA) was used to inhibit PP2Ac, which consists of a pool of three target-specific 19 to 25 nt siRNA. PP2A-Cα siRNA (h) is a pool of 3 different siRNA duplexes(5′ → 3′): A: Sense: GCAAAUCACCAGAUACAAAtt, Antisense: UUUGUAUCUGGUGAUUUGCtt; B: Sense: GAACUUGACGAUACUCUAAtt, Antisense: UUAGAGUAUCGUCAAGUUCtt; C: Sense: GGAUAGCAGCAAACAAUCAtt, Antisense: UGAUUGUUUGCUGCUAUCCtt. The nonsilencining control as a negative control consists of a scrambled sequence. For transfection, 2 × 105 cells were seeded in culture plates and transfected with PP2Ac siRNA or scrambled siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) for 48 h before treatment with JS-K for 24 h.
PP2A overexpression: Cells were trypsinized, collected by centrifugation, and resuspended in regular medium. A single-cell suspension was then seeded at 2 × 105 cells/well (12-well plates). Cells were infected or transfected with PP2A in serum-free medium the next day. For transfection, 2.5 μg of DNA was used for each transfection using lipofectamine 3000 reagent for 4 h. Then the infection medium was removed and replaced with complete medium for 48 h. Next, cells were treated with JS-K for 24 h to observe the expressions of proteins by Western blot analysis. Negative control plasmid was treated according the above methods.
Establishment of rat model of primary hepatic carcinoma
After one week of adaptive feeding, thirty-two male rats were randomly divided into four groups of eight each as follows: untreated control rats were received equivalent intraperitoneal injection of normal saline solution. The other rats were intraperitoneally injected with diethylnitrosamine (DEN, Sigma, USA) at 50 mg/kg body weight (bw), twice a week for four consecutive weeks. Then rats were received DEN at 50 mg/kg, once a week for another twelve consecutive weeks. JS-K treatment groups (0.25 mg/kg and 0.5 mg/kg) were given tail intravenous injection on the next day following DEN treatment, twice a week for 16 weeks. Following completion of treatment, the animals were sacrificed using an injection of chloral hydrate (400 mg/kg bw, i.p). After removing the livers, one liver fraction was frozen for further experiments, and another liver fraction was fixed in paraformaldehyde. All procedures were conducted according to our institutional guidelines for laboratory animals.
Immunohistochemical analysis
The fixed liver tissues were harvested and fixed for 24 h. Then tissue blocks were embedded in paraffin and sectioned at 3–4 μm thickness. The slides were baked overnight in oven at 60 °C, and were then washed in xylene and hydrated in different concentrations of alcohol. Subsequently, endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 20 min. To unmask the antigen, slides were submerged in citrate buffer (0.01 M, pH 6.0) at 95 °C for 20 min. After being blocked for 20 min with normal goat serum blocking solution, the sections were immunostained with the primary antibody at 4 °C overnight. The primary antisera were diluted 1:200 in PBS enriched with 0.3% Triton-X-100, 0.1% bovine serum albuminand 0.03% NaN3. Subsequently, the slides were then washed three times in PBS and visualized with 3,3-diaminobenzidine (DAB). Finally, the slides were counterstained with hematoxylin, dehydrated and mounted for imaging.
Western blot analysis
The protein of total cell lysis, mitochondria extracts were prepared according to the manufacturer’s instructions. Protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membrane (PVDF, Millipore,USA). The membranes were blocked with 5% nonfat milk in TBS-T (Tris-buffered saline and 1% Tween 20) and incubated with primary antibodies at 4 °C overnight. The dilution ratio of antibody was 1:1000. HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG were incubated for 2 h. The dilution ratio of the secondary antibody was 1:10000. Signals were visualized by Bio-Rad gel imaging and analysis system.
Statistical analysis
All results are presented as the means ± standard deviation from triplicate experiments performed in a parallel manner unless otherwise indicated. Statistical differences were evaluated by the Student’s t-Test and considered significant at the *P < 0.05 or **P < 0.01 level. All the figures shown in this article were obtained from at least three independent experiments.
Discussion
Uncontrolled tumor cell proliferation and escape from apoptosis play the most important role in human hepatocellular carcinoma growth. Therefore, the inhibition of cancer proliferation and apoptosis induction has been regarded as the crucial target for cancer treatment. In recent years, more researchers have focused on the effects of JS-K as a novel NO donor that was involved in multiple events in cancer, including inflammation, cell cycle progression, apoptosis, migration, and invasion [
13,
23,
24].
In the present study, JS-K exerted more cytotoxic effects and apoptosis induction in SMMC-7721 and HepG2 cells than PLC5, Huh-7 or Bel-7402 cells in a time- and dose-dependent manner. Moreover, JS-K hardly affected the viability of the non-tumor human hepatocyte L-02 cells. The levels of NO and the activity of PP2A were increased in SMMC-7721 and HepG2 cells following JS-K treatment, but not obviously in PLC5, Huh-7 or Bel-7402 cells, indicating that PP2A activation may be used as a novel anti-cancer target in JS-K-treated HCC cells. Thus, SMMC-7721 and HepG2 cells were selected as targeted cells in the subsequent experiment.
Apoptosis induction is a major means to eliminate cancer cells. The collapse of MMP is a typical apoptotic phenomenon in the functional impairment of mitochondria, which also facilitates the activation of caspases by regulating Bcl-2 family members on mitochondrial membrane [
25]. The members of Bcl-2 family proteins are composed of both pro- and anti-apoptotic proteins. The balance between pro- and anti-apoptotic proteins groups plays a pivotal role in inducing cell apoptosis and determining mitochondrial membrane integrity [
26]. Once the MMP has been disrupted, the mitochondrial permeability will be changed. Also, the pro-apoptotic signals lead to the release of Cyt c from the mitochondria into the cytosol. Cyt c subsequently activates caspase-9, which successively results in the activation of caspase-3 via cleavage induction [
27]. JS-K-treated sensitive HCC cells performed the typical morphological changes of apoptosis, i.e., nucleus condensation and nucleus fragmentation. Specially, JS-K significantly increased the proportion of cells with depolarized MMP, which implied the mitochondrial dysfunction as the early signs of apoptosis was involved in the sensitive HCC cells. Accordingly, JS-K also caused a significant increase of phosphatidylserine externalization in JS-K-treated cells. In addition, JS-K effectively initiated Bax/Bcl-2 modulation, Cyt c release and cleaved-caspase-9/3 activation. The expression of PARP as a downstream substrate of caspase-3 was increased whereas the expression of XIAP as an inhibitor of apoptosis was decreased. However, Z-VAD-FMK (a caspase inhibitor) treatment abolished the cleaved-caspase-9/3 activation, PARP cleavage, as well as the JS-K-induced decreases of XIAP. These results indicated that caspases successive activation is key regulators of the JS-K-induced apoptosis in both SMMC7721 cells and Bel-7402 cells. In addition, JS-K was found to cause dramatically an apparent elevation of NO with a dose-dependent manner using two different assays. Carboxy-PTIO as a NO scavenger significantly suppressed the apoptosis, reversed up-regulation of Bax, down-regulation of Bcl-2, the activation of cleaved-caspase-9/3 and PARP cleavage in the JS-K-treated cells. These results suggested that NO can be released from JS-K, which induced apoptosis through a caspase-mediated apoptotic pathway. Protein phosphorylation is regulated by a dynamic equilibrium between the protein kinases and phosphatases. Protein phosphatase 2A(PP2A), a serine/threonine phosphatase, regulates a variety of cellular processes, including cell proliferation, apoptosis and signal transduction [
28]. Accumulating evidence has been proven that PP2A as a tumor suppressor could reverse the actions of protein kinases by dephosphorylating PP2A substrates β-catenin, c-Myc, and Bcl-2 from threonine and serine residues of proteins [
29,
30]. Furthermore, PP2A is genetically inactivated in many types of cancer, including HCC cells, which is composed of a scaffolding A subunit, a variable regulatory B subunit, and a catalytic C subunit [
31]. The results revealed that PP2A-A and PP2A-B55 protein levels were unaffected by JS-K, but the levels of PP2A-C protein were raised in a dose-dependent manner. Simultaneously, incubation of sensitive HCC cells with JS-K caused to dephosphorylate the PP2A substrates, for example, β-catenin, c-Myc, and p-Bcl-2(Ser70).
The expression of β-catenin and c-Myc in the cytoplasm were significantly decreased, indicating the involvement of the Wnt/β-catenin signaling pathway. JS-K could modulate the Wnt/β-catenin/TCF-4 signaling pathway in Jurkat T-acute lymphoblastic leukemia cells [
13]. Abnormal activation of the Wnt/β-catenin signaling pathway facilitates human malignancies. After accumulation of β-catenin in the nucleus, it binds to the T-cell factor/lymphocyte enhancer factor (Tcf/lef) family of transcription factors activates its downstream responsive substrates such as c-Myc, cyclin D1 and survivin [
32]. These are related to the cell proliferation, apoptosis, cycle arrest and tumor metastasis in many human malignancies. c-Myc is induced on growth factor stimulation in normal cells and constitutively high in transformed cells. The c-Myc over expression is estimated to occur in 70% of human tumors. PP2A-mediated dephosphorylation c-Myc residue Ser62 enhances its degradation [
28]. It is also found that Bcl-2 is frequently overexpressed in human malignancies, and selective phosphorylation at Ser70 could enhance its anti-apoptotic activity. PP2A is a direct negative regulator of Bcl-2 via dephosphoralation of Bcl-2 at Ser 70 (p-S70-Bcl-2) [
33]. In support of PP2A acting as a activator, the results showed that knockdown of PP2A catalytic subunits impaired the PP2A-induced dephosphorylation of its downstream responsive substrates like Bcl-2 at Ser 70 whereas overexpression of PP2A has the opposite results, which demonstrated that PP2A activation was involved in JS-K-induced apoptosis of sensitive HCC cells. The addition of Carboxy-PTIO significantly abolished JS-K-induced PP2A-C activation and the substrates of PP2A inactivation such as β-catenin, c-Myc and p-Bcl-2(Ser70). Taking together, these results indicated that NO release from JS-K was related to the activation of PP2A that caused dephosphorylation of its substrates. Moreover, the anti-tumor effects of JS-K on rat model of primary hepatic carcinoma in vivo were further verified to be related to the activation of PP2A.
Notably, addition of okadaic acid (OA), a PP2A inhibitor, not only significantly abolished the effects of JS-K on the PP2A-C activation and the substrates of PP2A dephosphorylation, but also obviously protected against JS-K-induced apoptosis, such as Bax/Bcl-2 modulation and caspase successively activation in sensitive HCC cells. However, FTY720, a PP2A agonist, promoted JS-K-induced PP2A-C activation and strengthened the dephosphorylation of PP2A substrates, which further facilitated Bax/Bcl-2 modulation, cleaved caspase-9/3 activation and PARP cleavage resulting in the induction of apoptosis. These results probably implicated the involvement of the relationship between NO release from JS-K and PP2A activation.